Tubulysins, a novel class of antimitotic tetrapeptides that strongly inhibit microtubule polymerization, were first isolated from myxobacterial cultures (F. Sasse et al. J. Antibiot. 2000, 53, 879). Growth inhibition factors (GI50) ranging from nanomolar to picomolar concentrations, exceeding by 20- to 1000-fold that of well known chemotherapeutic agents like Taxol or Vinblastine, and a high cytotoxic activity on multidrug-resistant cell lines make tubulysins exciting leads for the development of novel anticancer drugs (H. Steinmetz at al., Angew. Chem., Int. Ed. 2004, 43, 4888).

Tubulysin D of formula (I) is the most potent member of this family of cytotoxins. Structurally, tubulysins are comprised of four amino acids which constitute the basic structure of this tetrapeptide as represented in formula (I): at the C-terminus tubuphenylalanine (Tup) or tubutyrosine (Tut), the complex tubuvaline (Tuv) as the central core, L-isoleucine (Ile), and the hydrophobic D-N-methyl pipecolic acid (Mep) as the N-terminal amino acid. An unusual amide N,O-acetal ester functionality which makes the middle part of the molecule extremely crowded, is also present. In general, natural tubulysins carrying this functionality have the greatest cytotoxic activity.
From the synthetic point of view this rare chemical motif represents a major challenge, and only few groups have succeeded on its introduction during the total synthesis of tubulysins (H. M. Peltier et al. J. Am. Chem. Soc. 2006, 128, 16019; and O. Pando et al. Org. Lett., 2009, 11, 5567). To date, several methods for the synthesis of tubulysins (DE 100 08 089, DE 102 54 439, DE 196 38 870, EP 1 562 979, EP 2 028 185, US 2005/0239713, DE 102 30 872, WO 2004/046170, WO 2009/055562) and analogues and/or conjugates of tubulysin (DE 102 30 874, DE 102 30 875, DE 103 05 531, DE 10 2004 030 227, US 2010/0048490, WO 2004/005269, WO 2004/005326, WO 2004/005327, WO 2008/138561, WO 2009/012958, WO 2009/134279, WO 2010/034724) have been proposed.
However, due to synthetic challenges, these methods generally provide the tubulysins and/or their analogues in relatively small amounts. In addition, extremely toxic electrophilic reagents have to be used during the preparation of the tubulysins and/or their analogues. Moreover, analogues of tubulysin which elude the N,O-acetal functionality, commonly exhibit inferior cytotoxic activities when compared with e.g. tubulysin D.